Article

Plant Molecular Biology Reporter

, 28:69

First online:

A 1-kb Bacteriophage Lambda Fragment Functions as an Insulator to Effectively Block Enhancer–Promoter Interactions in Arabidopsis thaliana

  • Stacy D. SingerAffiliated withUSDA-ARS Appalachian Fruit Research StationDepartment of Plant Pathology, New York State Agricultural Experiment Station, Cornell University
  • , Jean-Michel HilyAffiliated withUSDA-ARS Appalachian Fruit Research StationDepartment of Plant Pathology, New York State Agricultural Experiment Station, Cornell University
  • , Zongrang LiuAffiliated withUSDA-ARS Appalachian Fruit Research Station Email author 

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Abstract

Enhancers are known to be capable of overriding the specificity of nearby promoters in a distance-dependent manner, which is problematic when multiple promoters coexist in a single transgene unit. In an attempt to determine whether enhancer activation function is inversely related to its distance from the target promoter, we inserted 1-, 2-, and 4-kb bacteriophage λ fragments, respectively, between the cauliflower mosaic virus 35S enhancer and a flower-specific AGAMOUS second intron-derived promoter (AGIP) fused to the β-glucuronidase (GUS) coding region. In the absence of an insert sequence, the 35S enhancer activates AGIP-driven GUS expression in vegetative tissues of transgenic Arabidopsis thaliana lines. Moreover, neither the 2-kb nor the 4-kb λ fragment was able to block GUS expression in transgenic leaves, implying that the 35S enhancer can override a distance barrier of at least 4 kb in our system. Unexpectedly, insertion of the 1-kb λ insert into the same site resulted in diminished GUS expression in transgenic leaves. Our data indicate that this fragment functions as a true enhancer-blocking insulator that could potentially be utilized to minimize enhancer–promoter interference between multiple transcriptional units within a plasmid vector during plant transformation experiments.

Keywords

Arabidopsis thaliana Bacteriophage lambda Cauliflower mosaic virus 35S promoter Enhancer-blocking insulator Enhancer–promoter interaction